Tobramycin and Dexamethasone Ophthalmic Ointment »Tobramycin and Dexamethasone Ophthalmic Ointment contains not less than 90.0percent and not more than 120.0percent of the labeled amount of tobramycin (C18H37N5O9),and not less than 90.0percent and not more than 110.0percent of the labeled amount of dexamethasone (C22H29FO5). Packaging and storage— Preserve in collapsible ophthalmic ointment tubes. USP Reference standards á11ñ USP Dexamethasone RS.USP Tobramycin RS. Identification— A: To 1g of Ophthalmic Ointment in a test tube add 2mLof chloroform,and shake to dissolve.Add 0.5mLof sodium sulfate solution (1in 10),shake vigorously,and centrifuge:the clear supernatant aqueous liquid meets the requirements for Identificationtest Aunder Tobramycin.[NOTE—If,after centrifuging,an oily film remains on top of the supernatant aqueous liquid,transfer the supernatant aqueous liquid to a second test tube,and wash it with 2mLof chloroform.] B: The retention time of the major peak for dexamethasone in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay for dexamethasone. Sterility á71ñ It meets the requirements when tested as directed for Membrane Filtrationunder Test for Sterility of the Product to be Examined. Minimum fill á755ñ: meets the requirements. Water,Method Iá921ñ: not more than 1.0%,20mLof a mixture of toluene and methanol (7:3)being used in place of methanol in the titration vessel. Metal particles á751ñ: meets the requirements. Assay for tobramycin— Mobile phase ,2,4-Dinitrofluorobenzene reagent,Tris(hydroxymethyl)aminomethane reagent,Standard preparation,Resolution solution,and Chromatographic system—Proceed as directed in the Assayunder Tobramycin. Assay preparation— Transfer an accurately weighed portion of Ophthalmic Ointment,equivalent to about 4.5mg of tobramycin,to a separator,add 50mLof ether,and extract with four 20-to 25-mLportions of water.Combine the water extracts in a 100-mLvolumetric flask,dilute with water to volume,and mix. Derivatization procedure— Proceed as directed in the Assayunder Tobramycin,except to use 15.0mLof Assay preparationinstead of 4.0mL. Procedure— Proceed as directed in the Assayunder Tobramycin.Calculate the quantity of tobramycin (C18H37N5O9),in mg,in the portion of Ophthalmic Ointment taken by the formula: (4/150)(CE)(rU/rS), in which the terms are as defined therein. Assay for dexamethasone— Diluent— Prepare a mixture of methanol and water (750:250). Mobile phase— Prepare a suitable mixture of methanol and water (55:45),pass through a suitable filter having a 1-µm or finer porosity,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ). Standard preparation— Dissolve an accurately weighed quantity of USP Dexamethasone RSin Diluentto obtain a stock solution having a known concentration of about 0.2mg per mL.Transfer 15.0mLof this stock solution to a separator containing about 50mLof n-hexane,and shake.Allow the layers to separate,and drain the lower phase into a 50-mLvolumetric flask.Repeat the extraction with two 15-mLportions of Diluent,combining the lower phase from each extraction in the same 50-mLvolumetric flask.Dilute with Diluentto volume,and mix.This solution contains about 0.06mg of USP Dexamethasone RSper mL. Resolution solution— Prepare a stock solution of chlorobutanol and USP Dexamethasone RSin Diluentcontaining about 1mg of anhydrous chlorobutanol and 0.2mg of USP Dexamethasone RSper mL.Proceed as directed for Standard preparationbeginning with “Transfer 15.0mLof this stock solution to a separator.”The solution so obtained contains about 0.3mg of anhydrous chlorobutanol and 0.06mg of USP Dexamethasone RSper mL. Assay preparation— Transfer an accurately weighed quantity of Ophthalmic Ointment,equivalent to about 3mg of dexamethasone,to a separator containing about 50mLof n-hexane,and shake.Add 15mLof Diluent,and shake.Allow the layers to separate,and drain the lower phase into a 50-mLvolumetric flask.Repeat the extraction with two 15-mLportions of Diluent,combining the lower phase from each extraction in the same 50-mLvolumetric flask.Dilute with Diluentto volume,mix,and centrifuge.Use the clear solution. Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 206-nm detector and an 8.0-mm ×10-cm column that contains packing L1.The flow rate is about 3mLper minute.Chromatograph the Resolution solution,and measure the peak responses as directed for Procedure:the relative retention times are about 0.7for chlorobutanol and 1.0for dexamethasone;and the resolution,R,between chlorobutanol and dexamethasone is not less than 1.8.Chromatograph the Standard preparation,and measure the peak responses as directed for Procedure:the tailing factor is not more than 2;the column efficiency is not less than 350theoretical plates;and the relative standard deviation for replicate injections is not more than 2.0%. Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of dexamethasone (C22H29FO5)in the portion of Ophthalmic Ointment taken by the formula: 50C(rU/rS), in which Cis the concentration,in mg per mL,of USP Dexamethasone RSin the Standard preparation;and rUand rSare the dexamethasone peak responses obtained from the Assay preparationand the Standard preparation,respectively. Auxiliary Information— Staff Liaison:William W.Wright,Ph.D.,Scientific Fellow Expert Committee:(PA7)Pharmaceutical Analysis 7 USP28–NF23Page 1942 Phone Number:1-301-816-8335